Preparation method for human fibrinogen, and product

Abstract

A preparation method for human fibrinogen, in which a component I precipitate is used as a raw material, a specific solvent is used in combination with a low-temperature ethanol method to prepare a human fibrinogen bulk solution, and virus inactivation is performed by combining an S / D virus inactivation method and a dry heat virus inactivation method, thereby further guaranteeing the safety of the human fibrinogen. The prepared human fibrinogen has high purity and high reconstitution speed, a reconstitution solution is clear and only slightly opalescent, and the coagulation time is short.

Description

A method for preparing human fibrinogen and the product thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of protein production, and particularly relates to a method for preparing human fibrinogen and the product thereof. BACKGROUND

[0002] Human fibrinogen (Fg) is a protein component in plasma, with a content of 2-4 g, and plays a key role in the human blood clotting system. The last stage of blood clotting is the conversion of Fg into fibrin under the action of thrombin, and the fibrin and other blood cell components aggregate into insoluble masses to achieve the purpose of hemostasis. In addition to directly participating in the blood clotting process, Fg also plays an important role in platelet aggregation and blood viscosity. Common diseases related to Fg deficiency include congenital fibrinogen deficiency or deficiency, severe liver damage, cirrhosis, disseminated intravascular coagulation, postpartum hemorrhage, and coagulopathy caused by fibrinogen deficiency due to major surgery, trauma, or internal bleeding.

[0003] At present, the raw materials for extracting fibrin mainly include two methods: one is to use cold precipitate as raw material, and to obtain it through extraction, adsorption, centrifugation, chromatography and other manufacturing processes. The human fibrinogen precipitate obtained by using cold precipitate as raw material through extraction, adsorption, centrifugation, chromatography and other manufacturing processes has low solubility and is difficult to dissolve. The other is to use component I precipitate produced by low-temperature ethanol process, but this process mainly has the problem of low purity, and the purity of the product produced is below 80%, the product has a high impurity content, and the patient is prone to adverse reactions when injecting the product. On the other hand, the product of this process has a long reconstitution time, and since the product also contains a small amount of fibrous binding protein, the fibrous binding protein has poor thermal stability, especially when dry heat inactivation, it is easy to denature, which leads to the precipitation of protein during reconstitution.

[0004] Patent CN108017710B discloses a method for preparing human fibrinogen by double chromatography, which includes component I precipitate dissolution, filtration; S / D virus inactivation; Q Sepharose Fast Flow anion exchange chromatography; primary ultrafiltration concentration; heparin affinity chromatography; secondary ultrafiltration concentration; detection and preparation; split freezing and drying; cap rolling and dry heat inactivation. The protein content of the prepared product is 2.2-2.4%, and glycine and sodium citrate are used as protective agents. The purity of the human fibrinogen prepared by this method is improved to 96.2%, and the reconstitution time is 9 min. However, the effect of improving the stability of the product has not been involved.

[0005] In view of the current preparation process and clinical application status of human fibrinogen on the market, it is particularly necessary to develop a new human fibrinogen extraction process with high purity, good solubility and good stability. Technical problems

[0006] In order to solve the above problems, the present application provides a preparation method of human fibrinogen and the product. The preparation method provided by the present application uses component I precipitate as raw material, and obtains human fibrinogen stock solution by using a specific dissolving solution combined with low-temperature ethanol method. The preparation method provided by the present application combines S / D virus inactivation method and dry heat virus inactivation method to inactivate viruses, which can further ensure the safety of human fibrinogen. The human fibrinogen prepared by the preparation method provided by the present application has a purity of 87.4%-98.3%. The reconstitution speed is fast, and only 4-5 minutes is needed for reconstitution. The reconstitution solution is clear and only slightly milky.

[0007] In order to achieve the above-mentioned purposes, the technical solutions of the present application are as follows:

[0008] In one aspect, the present application provides a preparation method of human fibrinogen, characterized in that the preparation method comprises the following steps:

[0009] S1, filtering after dissolving component I precipitate to obtain filtrate A;

[0010] S2, inactivating filtrate A by S / D to obtain inactivated liquid;

[0011] S3, preparing human fibrinogen stock solution by twice low-temperature ethanol precipitation of inactivated liquid.

[0012] Specifically, the preparation method of component I precipitate in step S1 is as follows: obtaining component I precipitate by low-temperature ethanol precipitation method after removing cold precipitate of plasma.

[0013] Further specifically, the removing of cold precipitate comprises: first immersing the raw material plasma in 75% alcohol for 2-3 minutes, then rinsing the surface with water for injection, then conveying the raw material plasma out of the bag to a plasma melting tank, and performing interlayer circulation melting of the plasma with 10-37℃ water. After melting, stop the circulation, control the temperature of the plasma between 0-4℃, centrifugally separate the cold precipitate, and convey the plasma after removing the cold precipitate to a reaction tank for extraction and separation.

[0014] Further specifically, the low-temperature ethanol precipitation method comprises adding a buffer solution to adjust the pH of the plasma after removing the cold precipitate, adding low-temperature ethanol, and obtaining component I precipitate after centrifugation.

[0015] Preferably, the temperature of the plasma after removing the cold precipitate is controlled between 0-4℃.

[0016] Preferably, the low-temperature ethanol is 90%-95% ethanol at-15 to-20℃.

[0017] Further preferably, the low-temperature ethanol is 95% ethanol at -15°C.

[0018] Preferably, the volume ratio concentration of the ethanol to the plasma is 10%, and the flow rate of the added ethanol is not greater than 1.0 L / min.

[0019] Further preferably, the flow rate of the added ethanol includes 0.5 L / min, 0.6 L / min, 0.7 L / min, 0.8 L / min, 0.9 L / min, and 1.0 L / min.

[0020] Preferably, the pH adjustment is adjusting the pH to 6.8-7.2 by adding an acetic acid buffer solution with a pH of 4.0.

[0021] Specifically, the dissolving solution used in the dissolving in step S1 is dissolving solution A, and the dissolving solution A contains 1.6%-2.0% sodium citrate and 0.9%-1.1% sodium chloride.

[0022] Preferably, the dissolving solution A contains 1.85% sodium citrate and 0.9% sodium chloride.

[0023] Preferably, the preparation method of the dissolving solution A is dissolving sodium citrate and sodium chloride in water for injection, stirring uniformly, and adjusting the pH to 6.6-7.0 by adding 1 mol / L HCl.

[0024] Specifically, the ratio of the component I precipitate to the dissolving solution A in step S1 is 1:15-20 w / v.

[0025] Preferably, the ratio of the component I precipitate to the dissolving solution A in step S1 is 1:15 w / v.

[0026] Specifically, the filtration in step S1 is filtration using a plate-and-frame filter.

[0027] Preferably, the equilibration solution of the plate-and-frame filter is equilibration solution A, and the equilibration solution A contains 3.2%-4.0% calcium chloride and 0.9%-1.1% sodium chloride.

[0028] Further preferably, the filter plate used by the plate-and-frame filter is a K700 filter plate.

[0029] Further preferably, the equilibration solution of the plate-and-frame filter is equilibration solution A, and the equilibration solution A contains 3.7% calcium chloride and 0.9% sodium chloride.

[0030] Further preferably, the equilibration solution A, and the preparation method of the equilibration solution A is dissolving calcium chloride and sodium chloride in water for injection, stirring uniformly, and adjusting the pH to 6.6-7.0 by adding 1 mol / L HCl.

[0031] Further preferably, the ratio of sodium citrate in the dissolving solution A to calcium chloride in the balancing solution A is 1:1-3.

[0032] Still further preferably, the ratio of sodium citrate in the dissolving solution A to calcium chloride in the balancing solution A is 1:2.

[0033] Specifically, the flow rate of the filtration in step S1 is not more than 1.5 L / min.

[0034] Preferably, the flow rate of the filtration in step S1 is 1.0-1.5 L / min.

[0035] Specifically, the S / D inactivation in step S2 is carried out by adding S / D inactivation solution to the filtrate A at a ratio of 100 mL of S / D inactivation solution per liter of filtrate.

[0036] Preferably, the inactivation is carried out at a temperature of 24℃ for 6 h.

[0037] Specifically, the two low-temperature ethanol precipitations in step S3 include:

[0038] (1) adding ethanol to obtain a first precipitation, dissolving the first precipitation with dissolving solution B, filtering, and collecting the filtrate; and (2) adding ethanol to the filtrate to obtain a second precipitation, dissolving the second precipitation with dissolving solution C, filtering, and obtaining the human fibrinogen stock solution.

[0039] Preferably, the dissolving solution B contains 0.85%-1.2% sodium citrate, 0.9%-1.1% sodium chloride, and 0.35%-0.4% arginine hydrochloride.

[0040] Further preferably, the dissolving solution B contains 1.15% sodium citrate, 0.9% sodium chloride, and 0.37% arginine hydrochloride.

[0041] Preferably, the preparation method of the dissolving solution B is dissolving sodium citrate, sodium chloride, and arginine hydrochloride in water for injection, stirring until uniform, and adding glycine-hydrochloric acid buffer with pH = 3.6 to adjust the pH to 6.6-7.0.

[0042] Preferably, the ratio of the first precipitation to the dissolving solution B is 1:15-20 w / v.

[0043] Further preferably, the ratio of the first precipitation to the dissolving solution B is 1:15 w / v.

[0044] Preferably, the ethanol in step S3(1) is 45%-55% ethanol at -15 to -20℃.

[0045] Further preferably, the low-temperature ethanol is 50% ethanol at -15°C.

[0046] Further preferably, the volume ratio concentration of the ethanol to the plasma is 10%, and the flow rate of the added ethanol is not more than 1.0 L / min.

[0047] Preferably, the dissolving solution C contains 1.35%-1.75% sodium citrate and 3.5%-4.0% arginine hydrochloride.

[0048] Further preferably, the dissolving solution C contains 1.5% sodium citrate and 3.7% arginine hydrochloride.

[0049] Preferably, the preparation method of the dissolving solution C is to dissolve sodium citrate and arginine hydrochloride in water for injection, then stir uniformly, and add 1 mol / L HCl to adjust the pH to 6.8-7.0.

[0050] Preferably, the ratio of the second precipitation to the dissolving solution C is 1:5-6 w / v.

[0051] Further preferably, the ratio of the second precipitation to the dissolving solution C is 1:5 w / v.

[0052] Preferably, the filtration in step S3(1) is filtration using a plate-and-frame filter.

[0053] Further preferably, the filter plate used by the plate-and-frame filter is a SUPRA EK 1P filter plate.

[0054] Further preferably, the filter is equilibrated with the dissolving solution B.

[0055] Preferably, the ethanol in step S3(2) is 45%-55% ethanol at -15 to -20°C.

[0056] Further preferably, the low-temperature ethanol is 50% ethanol at -15°C.

[0057] Further preferably, the volume ratio concentration of the ethanol to the plasma is 10%, and the flow rate of the added ethanol is not more than 1.0 L / min.

[0058] Preferably, the filtration in step S3(2) is filtration using a plate-and-frame filter.

[0059] Further preferably, the filter plate used by the plate-and-frame filter is a SUPRA EK 1P filter plate.

[0060] Further preferably, the filter is equilibrated with the dissolving solution C.

[0061] Preferably, the flow rate of the filtration in steps S3 (1) and (2) is not more than 1.5 L / min.

[0062] Further preferably, the flow rate of the filtration in steps S3 (1) and (2) is 1.0-1.5 L / min.

[0063] Specifically, the preparation method further comprises:

[0064] S4, after adjusting the protein concentration, filtering, dispensing, freeze-drying, and capping to obtain human fibrinogen freeze-dried powder;

[0065] S5, the human fibrinogen freeze-dried powder is inactivated by a dry heat method to obtain a human fibrinogen preparation finished product.

[0066] Preferably, the solution used for adjusting the protein concentration in step S4 is solution C.

[0067] Preferably, the protein concentration adjustment in step S4 is adjusting the protein concentration to 2.5%-3.5%.

[0068] Preferably, the filtration in step S4 is filtration using a 0.45 μm filter.

[0069] Preferably, the freeze-drying process in step S4 is as follows:

[0070] The product is placed in a cabinet at room temperature, reduced to -6℃, and pre-frozen for 180 minutes; reduced to -40℃, and frozen for 180 minutes; increased to -15℃, and annealed for 120 minutes; reduced to -45℃, and continuously frozen for 360 minutes; the vacuum degree is started to be extracted to 10 Pa, and maintained at 10 Pa±5 Pa; increased to -40℃, and maintained for 60 minutes; increased to -15℃, and maintained for 120 minutes; increased to 0℃, and sublimed at low temperature, and maintained for 2220 minutes; increased to 10℃, and vacuum dried, and maintained for 120 minutes; increased to 33℃, and vacuum dried, and maintained for 240 minutes; the limit vacuum is extracted, and maintained at 33℃ until the vacuum is qualified, and the product temperature in the freeze-drying process is not more than 35℃.

[0071] Preferably, the dry heat method inactivation of viruses in step S5 is inactivation at a temperature of 99.5±5℃ for 30 min.

[0072] Further preferably, the dry heat method inactivation of viruses in step S5 is inactivation at a temperature of 100℃ for 30 min.

[0073] In another aspect, the present application provides a human fibrinogen prepared by the above preparation method.

[0074] The present application has the following beneficial effects:

[0075] (1) The preparation method provided by the present application prepares human fibrinogen with a purity of 87.4%-98.3%.

[0076] (2) The preparation method provided by the present application prepares human fibrinogen with a rapid reconstitution speed, which only needs 4-5 minutes to be reconstituted, and the reconstituted solution is clear and only slightly milky. The coagulation time is short, only 19-22 seconds. DETAILED DESCRIPTION

[0077] The technical solutions of the present application will be clearly and completely described below in combination with specific embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative efforts fall within the protection scope of the present application.

[0078] The preparation method of the base solution used in the present application is as follows:

[0079] 1. Acetic acid buffer solution with pH 4.0: take 65.6 g of anhydrous sodium acetate, add glacial acetic acid 244.9 mL, fully dissolve with an appropriate amount of water for injection, add water for injection to 1 L, and mix evenly;

[0080] 2. Glycine-hydrochloric acid buffer solution with pH = 3.6: take 250 mL of 0.2 mol / L glycine, add 25 mL of 0.2 mol / L HCl, and dilute to 1 L with water for injection.

[0081] 3. Preparation of S / D inactivation solution: the S / D inactivation solution contains 10% Tween 80 and 3.3% tributyl phosphate (TNBP); when preparing, first dissolve Tween 80 with warm water for injection, then add TNBP after it is completely dissolved, at this time the inactivation solution will become clear, the temperature is controlled at 25℃, inactivated for 8 hours, and the S / D inactivation solution is obtained.

[0082] Example 1

[0083] 1. Preparation of the dissolving solution:

[0084] Preparation of the dissolving solution A: the dissolving solution A contains 1.85% sodium citrate and 0.9% sodium chloride. The preparation method of the dissolving solution A is to dissolve sodium citrate and sodium chloride in water for injection, stir evenly, and add 1 mol / L HCl to adjust the pH to 6.6-7.0.

[0085] Preparation of the balancing solution A: the balancing solution A contains 3.7% calcium chloride and 0.9% sodium chloride. The preparation method of the balancing solution A is to dissolve calcium chloride and sodium chloride in water for injection, stir evenly, and add 1 mol / L HCl to adjust the pH to 6.6-7.0.

[0086] Preparation of dissolving solution B: Dissolving solution B contains 1.15% sodium citrate, 0.9% sodium chloride, 0.37% arginine hydrochloride. The preparation method of dissolving solution B is to dissolve sodium citrate, sodium chloride, arginine hydrochloride in water for injection, stir uniformly, and add glycine-hydrochloric acid buffer solution with pH = 3.6 to adjust the pH to 6.6-7.0.

[0087] Preparation of dissolving solution C: Dissolving solution C contains 1.5% sodium citrate, 3.7% arginine hydrochloride. The preparation method of dissolving solution C is to dissolve sodium citrate, arginine hydrochloride in water for injection, stir uniformly, and add 1 mol / L HCl to adjust the pH to 6.8-7.0.

[0088] 2. Preparation method of human fibrinogen

[0089] The raw plasma collection and testing are strictly in accordance with the provisions of "Human Plasma for Blood Product Production" in Chinese Pharmacopoeia 2020 Edition Part Three and the requirements of "Technical Guidance Principles for Implementation of Raw Material Plasma Quarantine Period Management".

[0090] (1) The raw plasma is first soaked in 75% alcohol for 2-3 min, then washed with water for injection on the surface, then the raw plasma is broken bag and transported to the plasma melting tank, and the interlayer is circulated and melted with 10-37℃ water. After melting, stop circulation, control the temperature of the plasma between 0-4℃, centrifugal separation of cold precipitate, and transport the plasma after removing the cold precipitate to the reaction tank for extraction and separation;

[0091] (2) Preparation of component I precipitate: the temperature of the plasma is controlled between 0 and 4℃, the pH is adjusted to 6.8-7.2 by adding acetic acid buffer solution with pH 4.0, the volume ratio concentration of ethanol to plasma is 10% by adding 95% ethanol at-15℃, the flow rate of ethanol added is 1L / min, and the final temperature of the reaction solution is controlled at-1 to-3℃. After adding ethanol, continuous centrifugation at 10000 rpm is performed to obtain component I precipitate;

[0092] (3) Dissolution and filtration of component I precipitate: according to the ratio of component I precipitate:dissolving solution A = 1:15 (w / v), dissolving solution I is added to component I precipitate, the temperature is controlled at 30-37℃, and stirring for 1 hour to make component I precipitate completely dissolved to obtain component I precipitate dissolving solution. Install K700 filter plate, use equilibrium liquid A to balance the filter, balance for 20 min, then start filtering component I precipitate dissolving solution, the flow rate is 1.5L / min, and obtain filtrate A;

[0093] (4) S / D inactivation: according to the ratio of 100mL S / D inactivation solution per liter of filtrate, add S / D inactivation solution to filtrate A at a speed of 0.5L / min under stirring, the temperature is maintained at 24℃, inactivate for 6h, and obtain inactivated solution;

[0094] (5) First precipitation: the inactivation solution is cooled to 0-1°C, 50% ethanol at -15°C is added, the volume ratio concentration of ethanol to plasma is 10%, the flow rate of adding ethanol is 1 L / min, the final temperature of the reaction solution is controlled at -1 to -3°C, and after the addition of ethanol is completed, continuous centrifugation at 10000 rpm is performed to obtain a first precipitate;

[0095] (6) First precipitate dissolution and filtration: according to the ratio of first precipitate: dissolution solution B = 1:15 (w / v), dissolution solution B is added to the first precipitate, stirring is performed for 1 hour, the temperature is controlled at 30-37°C, the first precipitate is completely dissolved to obtain a first precipitate dissolution solution. A SUPRA EK 1P filter plate is installed, the filter is equilibrated with dissolution solution B, after equilibration for 20 min, the first precipitate dissolution solution is filtered, the flow rate is 1.5 L / min, and filtrate B is obtained;

[0096] (7) Second precipitation: the filtrate B is cooled to 0-1°C, 50% ethanol at -15°C is added, the volume ratio concentration of ethanol to plasma is 10%, the flow rate of adding ethanol is 1 L / min, the final temperature of the reaction solution is controlled at -1 to -3°C, and after the addition of ethanol is completed, continuous centrifugation at 10000 rpm is performed to obtain a second precipitate;

[0097] (8) Preparation of human fibrinogen stock solution: according to the ratio of second precipitate: dissolution solution C = 1:5 (w / v), dissolution solution C is added to the second precipitate, stirring is performed for 30 min, the temperature is controlled at 30-37°C, the second precipitate is completely dissolved to obtain a second precipitate dissolution solution. A SUPRA EK 1P filter plate is installed, the filter is equilibrated with dissolution solution C, after equilibration for 20 min, the second precipitate dissolution solution is filtered, the flow rate is 1.5 L / min, and a human fibrinogen stock solution is obtained;

[0098] (9) Liquid preparation: the human fibrinogen stock solution is adjusted to a protein concentration of 2.5% using dissolution solution C, and after filtration using a 0.45 μm filter core, a liquid preparation semi-finished product is prepared;

[0099] (10) Sub-packing and freeze-drying: the liquid preparation semi-finished product is sterilized and sub-packed into 25 mL injection vials, freeze-dried, capped after freeze-drying, and a human fibrinogen freeze-dried powder is prepared;

[0100] (11) Dry heat method for inactivating viruses: the human fibrinogen freeze-dried powder is placed in a dry heat sterilizer, inactivated at 100°C for 30 min, and a human fibrinogen preparation finished product is obtained.

[0101] Example 2

[0102] 1. Preparation of dissolution solution:

[0103] Preparation of dissolving solution A: Dissolving solution A contains 1.6% sodium citrate, 1.1% sodium chloride. The preparation method of dissolving solution A is to dissolve sodium citrate, sodium chloride in water for injection, stir uniformly, and adjust pH to 6.6-7.0 by adding 1 mol / L HCl.

[0104] Preparation of equilibrium solution A: Equilibrium solution A contains 3.2% calcium chloride and 1.1% sodium chloride. The preparation method of equilibrium solution A is to dissolve calcium chloride and sodium chloride in water for injection, stir uniformly, and adjust pH to 6.6-7.0 by adding 1 mol / L HCl.

[0105] Preparation of dissolving solution B: Dissolving solution B contains 1.2% sodium citrate, 1.1% sodium chloride, 0.35% arginine hydrochloride. The preparation method of dissolving solution B is to dissolve sodium citrate, sodium chloride, and arginine hydrochloride in water for injection, stir uniformly, and adjust pH to 6.6-7.0 by adding glycine-hydrochloric acid buffer with pH = 3.6.

[0106] Preparation of dissolving solution C: Dissolving solution C contains 1.35% sodium citrate and 3.5% arginine hydrochloride. The preparation method of dissolving solution C is to dissolve sodium citrate and arginine hydrochloride in water for injection, stir uniformly, and adjust pH to 6.8-7.0 by adding 1 mol / L HCl.

[0107] 2. Preparation method of human fibrinogen

[0108] The raw plasma collection and testing are strictly in accordance with the provisions of "Human Plasma for Blood Product Production" in Chinese Pharmacopoeia 2020 Edition Part Three and the requirements of "Technical Guidance Principles for Implementation of Raw Material Plasma Quarantine Period Management".

[0109] (1) First, immerse the raw plasma in 75% alcohol for 2-3 minutes, then rinse the surface with water for injection, then transport the raw plasma out of the bag to the plasma melting tank, and perform interlayer circulation melting with 10-37℃ water. Stop circulation after melting, control the plasma temperature between 0-4℃, centrifuge the cold precipitate, and transport the plasma after removing the cold precipitate to the reaction tank for extraction and separation;

[0110] (2) Preparation of component I precipitate: control the plasma temperature between 0 and 4℃, adjust pH to 6.8-7.2 by adding pH 4.0 acetic acid buffer, add 95% ethanol at -15℃ to make the volume ratio concentration of ethanol and plasma 10%, the flow rate of added ethanol is 1L / min, and the final temperature of the reaction solution is controlled at -1 to -3℃, then continuously centrifuge at 10000 rpm after adding ethanol to obtain component I precipitate;

[0111] (3) Dissolution and filtration of component I precipitate: according to the ratio of component I precipitate: dissolution solution A = 1:20 (w / v), add dissolution solution I to the component I precipitate, control the temperature at 30-37°C, and stir for 1 hour to completely dissolve the component I precipitate, to obtain a component I precipitate dissolution solution. Install a K700 filter plate, use equilibrium solution A to balance the filter, and after 20 minutes of balancing, start filtering the component I precipitate dissolution solution at a flow rate of 1.5 L / min, to obtain a filtrate A;

[0112] (4) S / D inactivation: according to the ratio of adding 100 mL of S / D inactivation solution per liter of filtrate, add the S / D inactivation solution to the filtrate A at a speed of 0.5 L / min under stirring, maintain the temperature at 24°C, and inactivate for 6 hours, to obtain an inactivated solution;

[0113] (5) First precipitation: cool the inactivated solution to 0-1°C, add 50% ethanol at -15°C, so that the volume ratio concentration of ethanol to plasma is 10%, add the ethanol at a flow rate of 1 L / min, and control the final temperature of the reaction solution at -1 to -3°C, after adding the ethanol, continuously centrifuge at 10000 rpm, to obtain a first precipitate;

[0114] (6) Dissolution and filtration of the first precipitate: according to the ratio of the first precipitate: dissolution solution B = 1:20 (w / v), add dissolution solution B to the first precipitate, stir for 1 hour, and control the temperature at 30-37°C, to completely dissolve the first precipitate, to obtain a first precipitate dissolution solution. Install a SUPRA EK 1P filter plate, use dissolution solution B to balance the filter, and after 20 minutes of balancing, start filtering the first precipitate dissolution solution at a flow rate of 1.5 L / min, to obtain a filtrate B;

[0115] (7) Second precipitation: cool the filtrate B to 0-1°C, add 50% ethanol at -15°C, so that the volume ratio concentration of ethanol to plasma is 10%, add the ethanol at a flow rate of 1 L / min, and control the final temperature of the reaction solution at -1 to -3°C, after adding the ethanol, continuously centrifuge at 10000 rpm, to obtain a second precipitate;

[0116] (8) Preparation of human fibrinogen stock solution: according to the ratio of the second precipitate: dissolution solution C = 1:6 (w / v), add dissolution solution C to the second precipitate, stir for 30 minutes, and control the temperature at 30-37°C, to completely dissolve the second precipitate, to obtain a second precipitate dissolution solution. Install a SUPRA EK 1P filter plate, use dissolution solution C to balance the filter, and after 20 minutes of balancing, start filtering the second precipitate dissolution solution at a flow rate of 1.5 L / min, to obtain a human fibrinogen stock solution;

[0117] (9) Preparation of the solution: the human fibrinogen stock solution was adjusted to a protein concentration of 2.5% using the dissolving solution C, and the solution was filtered through a 0.45 μm filter to obtain a semi-finished product;

[0118] (10) Sub-packing and freeze-drying: the semi-finished product was sterilized and sub-packed into 25 mL injection vials, and then freeze-dried to obtain a human fibrinogen lyophilized powder;

[0119] (11) Virus inactivation by dry heat: the human fibrinogen lyophilized powder was placed in a dry heat sterilizer and inactivated at 100°C for 30 min to obtain a finished product of human fibrinogen preparation.

[0120] Example 3

[0121] Example 3 differs from Example 1 in that the preparation method of the dissolving solution is different.

[0122] Preparation of the dissolving solution A: the dissolving solution A contains 2.0% sodium citrate and 0.9% sodium chloride. The dissolving solution A is prepared by dissolving sodium citrate and sodium chloride in water for injection, stirring uniformly, and adjusting the pH to 6.6-7.0 by adding 1 mol / L HCl.

[0123] Preparation of the equilibration solution A: the equilibration solution A contains 4.0% calcium chloride and 0.9% sodium chloride. The equilibration solution A is prepared by dissolving calcium chloride and sodium chloride in water for injection, stirring uniformly, and adjusting the pH to 6.6-7.0 by adding 1 mol / L HCl.

[0124] Preparation of the dissolving solution B: the dissolving solution B contains 0.85% sodium citrate, 0.9% sodium chloride, and 0.4% arginine hydrochloride. The dissolving solution B is prepared by dissolving sodium citrate, sodium chloride, and arginine hydrochloride in water for injection, stirring uniformly, and adjusting the pH to 6.6-7.0 by adding a glycine-hydrochloric acid buffer with pH = 3.6.

[0125] Preparation of the dissolving solution C: the dissolving solution C contains 1.75% sodium citrate and 4.0% arginine hydrochloride. The dissolving solution C is prepared by dissolving sodium citrate and arginine hydrochloride in water for injection, stirring uniformly, and adjusting the pH to 6.8-7.0 by adding 1 mol / L HCl.

[0126] Comparative Example 1

[0127] Comparative Example 1 differs from Example 1 only in that the equilibration solution A in step (3) is replaced by the dissolving solution A.

[0128] Comparative Example 2

[0129] Comparative Example 2 differs from Example 1 only in that the preparation method of the dissolving solution B is different.

[0130] Preparation of dissolving solution B: dissolving solution B contains 1.2% sodium citrate, 1.1% sodium chloride, 0.35% arginine hydrochloride. The preparation method of dissolving solution B is to dissolve sodium citrate, sodium chloride, arginine hydrochloride in water for injection, stir uniformly, and adjust pH to 6.6-7.0 by adding 1 mol / L HCl.

[0131] Comparative Example 3

[0132] The difference between Comparative Example 3 and Example 1 is only that:

[0133] 1. The equilibrium solution A in step (3) is replaced by dissolving solution A.

[0134] 2. The preparation method of dissolving solution B is different.

[0135] Preparation of dissolving solution B: dissolving solution B contains 1.2% sodium citrate, 1.1% sodium chloride, 0.35% arginine hydrochloride. The preparation method of dissolving solution B is to dissolve sodium citrate, sodium chloride, arginine hydrochloride in water for injection, stir uniformly, and adjust pH to 6.6-7.0 by adding 1 mol / L HCl.

[0136] Comparative Example 4

[0137] The difference between Comparative Example 4 and Example 1 is only that: the preparation method of dissolving solution A is different.

[0138] Preparation of dissolving solution A: dissolving solution A contains 1.4% sodium citrate, 0.9% sodium chloride, 1.1% sucrose. The preparation method of dissolving solution A is to dissolve sodium citrate, sodium chloride, sucrose in water for injection, stir uniformly, and adjust pH to 6.6-7.0 by adding 1 mol / L HCl.

[0139] Comparative Example 5

[0140] The difference between Comparative Example 5 and Example 1 is only that: the preparation method of dissolving solution C is different.

[0141] Preparation of dissolving solution C: dissolving solution C contains 0.9% sodium citrate, 0.36% arginine hydrochloride. The preparation method of dissolving solution C is to dissolve sodium citrate, arginine hydrochloride in water for injection, stir uniformly, and adjust pH to 6.8-7.0 by adding 1 mol / L HCl.

[0142] Experimental Example 1

[0143] The pH, purity, coagulation activity, reconstitution time, visible foreign matter, stability, and moisture of Examples 1-3 and Comparative Examples 1-5 were determined. The determination method is as follows:

[0144] Determination of pH: refer to General 0631 in Chinese Pharmacopoeia (2020 edition).

[0145] Purity determination: refer to the first method of 2020 edition of Chinese Pharmacopoeia 0731.

[0146] Coagulant activity: the thrombin solution was diluted with 0.9% sodium chloride solution to 3 IU / mL, and the test sample solution was diluted to 3 mg / mL for standby. Add an appropriate amount of preheated thrombin solution (3 IU / mL) to the reaction tube at 37℃, and then add an equal amount of test sample solution (3 mg / mL), shake well. Place at 37℃ and record the coagulation time with an automatic detection instrument.

[0147] Reconstitution time determination: the test sample was equilibrated to 37℃, and 37℃ sterilized water for injection was added according to the labeled amount (25mL), and gently shaken, which should be completely dissolved within 30 minutes.

[0148] Visible foreign matter determination: refer to 2020 edition of Chinese Pharmacopoeia 0904.

[0149] Stability determination: after the test sample was reconstituted, it was placed in a 30-37℃ water bath for 60 minutes, and whether there were clots or fibrin precipitates was observed.

[0150] Moisture determination: refer to 2020 edition of Chinese Pharmacopoeia 0832.

[0151] The test results of examples 1-3 are shown in table 1, and the test results of comparative examples 1-5 are shown in table 2.

[0152] Table 1 Test results of examples 1-3

[0153]

[0154] Table 2 Test results of comparative examples 1-5

[0155]

[0156] The above detailed description is a specific description of one of the feasible embodiments of the present application, which is not used to limit the patent scope of the present application. It should be noted that any equivalent implementation or change made without departing from the present application shall be included in the scope of the technical solutions of the present application. Therefore, the protection scope of the present application patent should be subject to the appended claims.

Claims

1. A method for the preparation of human fibrinogen, characterized in that, The preparation method comprises the following steps: S1, dissolving the precipitated component I to obtain filtrate A; S2, the filtrate A is inactivated by S / D to obtain an inactivated liquid; S3, the inactivated liquid is prepared by twice low-temperature ethanol precipitation to obtain a human fibrinogen stock solution; The dissolving solution used in step S1 is dissolving solution A, and the dissolving solution A contains 1.6%-2.0% sodium citrate, 0.9%-1.1% sodium chloride; The filtration in step S1 is carried out by using a plate and frame filter, and the balance liquid of the plate and frame filter is balance liquid A, and the balance liquid A contains 3.2%-4.0% calcium chloride and 0.9%-1.1% sodium chloride; The twice low-temperature ethanol precipitation in step S3 comprises adding ethanol to obtain a first precipitate, dissolving the first precipitate with dissolving solution B, filtering, collecting the filtrate, adding ethanol to the filtrate to obtain a second precipitate, dissolving the second precipitate with dissolving solution C, filtering, and obtaining a human fibrinogen stock solution; The dissolving solution B contains 0.85%-1.2% sodium citrate, 0.9%-1.1% sodium chloride, and 0.35%-0.4% arginine hydrochloride; and the preparation method of the dissolving solution B comprises the following steps: dissolving sodium citrate, sodium chloride and arginine hydrochloride in water for injection, stirring uniformly, and adding glycine-hydrochloric acid buffer with pH=3.6 to adjust the pH; The dissolving solution C contains 1.35%-1.75% sodium citrate and 3.5%-4.0% arginine hydrochloride; The ratio of sodium citrate in the dissolving solution A to calcium chloride in the balance liquid A is 1:

2.

2. The production method according to claim 1, characterized by, The component I in step S2 The ratio of the precipitate to the dissolving solution A is 1:15-20 w / v.

3. The preparation method according to claim 1, characterized in that, The ratio of the first precipitate to the dissolving solution B in step S3 is 1:15-20 w / v.

4. The method of claim 1, wherein, The ratio of the second precipitate to the dissolving solution C in step S3 is 1:5-6 w / v.

5. The preparation method according to claim 1, characterized in that, The preparation method further comprises the following steps: S4, adjusting the protein concentration, filtering, dispensing, freeze-drying, capping, and obtaining human fibrinogen lyophilized powder; S5, the human fibrinogen lyophilized powder is inactivated by dry heat method to obtain human fibrinogen preparation finished product.

6. The production method according to claim 5, wherein The adjusting of the protein concentration in step S4 is adjusting the protein concentration to 2.5%-3.5% by using the dissolving solution C.

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